1. Field of the Invention
This invention relates to oligonucleotides that are complementary to the thioredoxin and thioredoxin reductase genes which modulate tumor cell growth in mammals. This invention is also related to methods of using such compounds in inhibiting the growth of tumor cells in mammals. This invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable excipient and an effective amount of a compound of this invention.
The following publications, patent applications and patents are cited in this application as superscript numbers:
1. Holmgren, xe2x80x9cEnzymatic reduction-oxidation of protein disulfide by thioredoxinxe2x80x9d Methods Enzymol 107:295-300 (1984)
2. Wright and Anazodo, xe2x80x9cAntisense Molecues and their potential for the treatment of cancer and AIDSxe2x80x9d The Cancer Journal 4:185-189 (1995)
3. Matthew et al., xe2x80x9cThioredoxin regulates the DNA binding activity of NF-Kappa B by reduction of a disulphide bond involving cysteine 62 Nucleic Acids Res. 20:3821-3830 (1992)
4. Bannister et al., xe2x80x9cIn vitro DNA binding activity of Fos/Jun and BZLF1 but not C/EBP is affected by redox changesxe2x80x9d Oncogene 6:1243-50 (1991)
5. Cromlish and Roeder xe2x80x9cHuman transcription factor IIIC (TFIIIC)xe2x80x9d J. Biol. Chem. 264:18100-9 (1989)
6. Abate et al., xe2x80x9cRedox regulation of fos and jun DNA-binding activity in vitroxe2x80x9d Science 249:1157-1161 (1990)
7. Tagaya et al., xe2x80x9cATL-derived factor (ADF), an IL-2 receptor/Tac inducer homologous to thioredoxin: possible involvement of dithiol-reduction in the Il-2 receptor induction EMBO J. 8:757-64 (1989)
8. Powis et al., xe2x80x9cThe thioredoxin/thioredoxin reductase redox system and control of cell growthxe2x80x9d Oncol. Res. 6:53944 (1994)
9. Oblong et al., xe2x80x9cSite directed mutagenesis of active site cysteines in human thioredoxin produces competitive inhibitors of human thioredoxin reductase and elimination of mitogenic properties of thioredoxin J. Biol. Chem. 269:11714-20 (1994)
10. Gasdaska et al. xe2x80x9cCell growth stimulation by the redox protein thioredoxin occurs by a novel helper mechanismxe2x80x9d Cell Growth Differ 6:1643-1650 (1995)
11. Gasdaska et al., xe2x80x9cThe predicted amino acid sequence of human thioredoxin is identical to that of the autocrine growth factor human adult T-cell derived factor (ADF); thioredoxin mRNA is elevated in some human tumorsxe2x80x9d Biochem Biophys. Acta 1218:292-296 (1994)
12. Berggren et al., xe2x80x9cThioredoxin and thioredoxin reductase gene expression in human tumors and cell lines and the effects of serum stimulation and hypoxiaxe2x80x9d Anticancer Res. 16:3459-3466 (1996)
13. Fujii et al., xe2x80x9cCoexpression of adult T-cell leukemia-derived factor, a human thioredoxin homologue and human papillomavirus DNA in neoplastic cervical squamous epitheliumxe2x80x9d Cancer 68:1583-91 (1991)
14. Kawahara et al., xe2x80x9cEnhanced coexpression of thioredoxin and high mobility group protein 1 genes in human hepatocellular carcinoma and the possible association with decreased sensitivity to cisplatinxe2x80x9d Cancer Res. 56:5330-3 (1996)
15. Gallegos et al., xe2x80x9cTransfection with human thioredoxin increases cell proliferation and a dominant-negative mutant thioredoxin reverses the transformed phenotype of human breast cancer cellsxe2x80x9d, Cancer Res. 56:5765-70 (1996)
16. Baker et al., xe2x80x9cThioredoxin, a gene found overexpressed in human cancer, inhibits apoptosis in vitro and in vivoxe2x80x9d Cancer Res. 57:5162-7 (1997)
17. Mau and Powis, xe2x80x9cInhibition of cellular thioredoxin reductase by diaziquinone and doxorubicin. Relationship to the inhibition of cell proliferation and decreased ribonucleotide reductase activityxe2x80x9d Biochem Pharmacol 43: 1621-7 (1992)
18. Mau and Powis, xe2x80x9cMechanism-based inhibition of thioredoxin reductase by antitumor quinoid compoundsxe2x80x9d Biochem Pharmacol 43: 1613-20 (1992)
19. Schallreuter and Wood, xe2x80x9cNew aspects in the pathophysiology of cutaneous melanoma: a review of the role of thioproteins and the effect to nitrosoureasxe2x80x9d Melanoma Res. 1:159-167 (1991)
20. Schallreuter and Wood, xe2x80x9cThe stereospecific suicide inhibition of human melanoma thioredoxin reductase by 13-cis-retinoic acidxe2x80x9d Biochem Biophys. Res. Commun. 160:573-9 (1989)
21. Curcio et al., xe2x80x9cOligonucleotides as modulators of cancer gene expressionxe2x80x9d Pharmacol Ther. 74:317-32 (1997)
22. Narayanan and Akhtar, xe2x80x9cAntisense therapyxe2x80x9d Curr Opin. Oncol. 8:509-15 (1996)
23. Remington""s Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa. 17th ed. (1985)
24. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989, 1992)
25. Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989)
26. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons, New York (1988)
27. Hurta and Wright, xe2x80x9cMalignant transformation by H-ras results in aberrant regulation of ribonucleotide reductase gene expression by transforming growth factor-betaxe2x80x9d J. Cell Biochem 57:543-556 (1995)
28. Tagaya et al., xe2x80x9cATL-derived factor (ADF) an IL-2-receptor/Tac inducer homologous to thioredoxin: possible involvement of dithiol-reduction in the IL-2 receptor inductionxe2x80x9d EMBO J. 13:2244 (1994)
29 Choy et al., xe2x80x9cMolecular mechanisms of drug resistance involving ribonucleotide reductase: hydroxyurea resistance in a series of clonally related mouse cell lines selected in the presence of increasing drug concentrationsxe2x80x9d Cancer Res. 48:2029-2035 (1988)
30. Fan et al., xe2x80x9cRibonucleotide reductase R2 component is a novel malignancy determinant that cooperates with activated oncogenes to determine transformation and malignant potentialxe2x80x9d Proc. Natl. Acad. Sci USA 93:14036-40 (1996)
31. Huang and Wright, xe2x80x9cFibroblast growth factor mediated alterations in drug resistance and evidence of gene amplificationxe2x80x9d Oncogene 9:491499 (1994)
32. Gasdaska et al., xe2x80x9cCloning and sequencing of a human thioredoxin reductasexe2x80x9d FEBS Letters 373:5-9 (1995)
29. Nielsen et al.; Science (1991) 354:1497
30. Good and Nielsen; xe2x80x9cInhibition of translation and bacterial growth by peptide nucleic acid targeted to ribosomal RNAxe2x80x9d, PNAS USA (1998) 95:2073-2076
31. Buchardt, deceased, et al., U.S. Pat. No. 5,766,855
32. Buchardt, deceased, et al., U.S. Pat. No. 5,719,262
33. U.S. Pat. No. 5,034,50633 
34. Altschul, et al. xe2x80x9cBasic local alignment search toolxe2x80x9d, J. Mol. Biol. (1990) 215:403-10;
35. Devereux J. et al., xe2x80x9cA comprehensive set of sequence analysis programs for the VAXxe2x80x9d, Nucleic Acids Res. (1984) 12:387-395;
36. Chang et al.36; Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995);
37. Vega et al.37; Gene Targeting, CRC Press, Ann Arbor, Mich. (1995)
38. Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston, Mass. (1988)
39. Sullivan 1994, U.S. Pat. No. 5,225,347
40. U.S. Pat. No. 5,023,252 issued Jun. 11, 1991
41. Felgner et al., U.S. Pat. No. 5,580,859
42. U.S. Pat. No. 5,011,472
43. Dreeley et al., Science, 258:1650-1654 (1992)
All of the above publications, patent applications and patents are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.
2. State of the Art
Thioredoxin is a small ubiquitous redox protein originally identified as a reducing cofactor for ribonucleotide reductase which is essential for DNA synthesis1. Thioredoxin and thioredoxin reductase comprise the thioredoxin system. Thioredoxin reductase is a selenocysteine containing flavoenzyme which uses NADPH as a proton donor to reduce thioredoxin which in turn reduces other proteins and therefore influences their functions.
In recent years, mammalian thioredoxin has been implicated in a variety of other biochemical pathways. For example, it modulates redox properties of transcription factors by dithiol disulfide exchanges, which alter their DNA binding characteristics. Transcription factors such as NF-xcexaB3, BZLFI4 and TFIIIC5 are directly regulated, while AP-1 activation is mediated indirectly through the nuclear redox factor Ref-1 which is further reduced by thioredoxin6. In addition, thioredoxin has been shown to facilitate refolding of disulfide-containing proteins, to activate the glucocorticoid or interleukin-2-receptors, to inhibit human immunodeficiency virus expression in macrophages, to reduce H2O2 scavenge free radicals, to protect cells against oxidative stress and to be an early pregnancy factor.
Cloned human thioredoxin has been shown to be similar to a growth factor termed adult T-cell leukemia-derived factor, released by HTLV-1 transformed T cells7. It utilizes a pathway for secretion. Extracellularly expressed thioredoxin stimulates the proliferation of normal fibroblasts, lymphoid cells and a number of human solid tumor cell lines8 and 9. Redox-inactive forms have been used to show that the growth stimulation requires a redox activity of thioredoxin9. The growth stimulation by thioredoxin appears to be induced indirectly through sensitizing cells to other growth factors10. Subsequently, thioredoxin has been reported to be over-expressed in some primary tumors such as lung, colon, cervical and hepatocellular carcinoma11-14. Furthermore, human breast cancer cells transfected with wild-type thioredoxin cDNA have shown increased tumor growth15, decreased spontaneous apoptosis in vivo16 and decreased sensitivity to apoptosis induced by a variety of anticancer therapeutic compounds16. On the other hand, cells transfected with dominant-negative, redox-inactive mutant thioredoxin have shown reduced anchorage-independent growth in vitro and inhibition of tumor growth in vivo15.
Thioredoxin reductase has been shown to be overexpressed by a number of human tumors12. Inhibition of cellular thioredoxin reductase by antitumor quinones17 and 18, nitrosoureas19 and 13-cis-retinoic acid20 have led to a decreased activity of the thioredoxin system and consequent contribution to the growth inhibitory activity.
Antisense oligonucleotides have been utilized to inhibit gene expression in a target-specific manner by sequence-specific hybridization to target mRNA2. Antisense oligonucleotide-mediated repression of oncogenes has revealed that these compounds may be useful for identifying mechanisms governing oncogenesis21 and may also be promising as novel therapeutic compounds for the treatment of cancer22. Therefore, it would be desirable to identify antisense oligonucleotides directed against thioredoxin and thioredoxin reductase which act to inhibit the expression of thioredoxin or thioredoxin reductase with higher specificity and with less toxicity.
This invention is directed to antisense oligonucleotides which modulate the expression of the thioredoxin and ihioredoxin reductase genes in tumor cells in mammals and pharmaceutical compositions comprising such antisense oligonucleotides. This invention is also related to methods of using such antisense oligonucleotides for inhibiting the growth and metastasis of tumor cells in mammals.
Accordingly, in one of its composition aspects, this invention is directed to an antisense oligonucleotide, which oligonucleotide comprises from about 3 to about 50 nucleotides, which nucleotides are complementary to the thioredoxin mRNA or the thioredoxin reductase mRNA of a mammal. The antisense oligonucleotide may be nuclease resistant and may have one or more phosphorothioate internucleotide linkages. The antisense oligonucleotide may further comprise additional nucleotides which are not complementary to the thioredoxin mRNA or the thioredoxin reductase mRNA.
In another of its composition aspects, this invention is directed to an antisense oligonucleotide comprising from about 17 to about 50 nucleotides, wherein the oligonucleotide comprises a sequence selected from the group consisting of sequences 2601-2626 [SEQ ID NOs:1-26] as set forth in Table 1.
In another of its composition aspects, this invention is directed to an antisense oligonucleotide comprising from about 20 to about 50 nucleotides, wherein the oligonucleotide comprises a sequence selected from the group consisting of sequences 3001-3040 [SEQ ID NOs:27-66] as set forth in Table 2.
In still another of its composition aspects, this invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of an antisense oligonucleotide, which oligonucleotide comprises from about 3 to about 50 nucleotides, which nucleotides are complementary to the thioredoxin gene or the thioredoxin reductase gene of a mammal.
In still another of its composition aspects, this invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of an antisense oligonucleotide comprising from about 17 to about 50 nucleotides, wherein the oligonucleotide comprises a sequence selected from the group consisting of sequences 2601-2626 [SEQ ID NOs:1-26] as set forth in Table 1.
In still another of its composition aspects, this invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of an antisense oligonucleotide comprising from about 20 to about 50 nucleotides, wherein the oligonucleotide comprises a sequence selected from the group consisting of sequences 3001-3040 [SEQ ID NOs:27-66] as set forth in Table 2.
In one of its method aspects, this invention is directed to a method for inhibiting the growth of a mammalian tumor comprising, administering to a mammal suspected of having the tumor an effective amount of an antisense oligonucleotide comprising from about 3 nucleotides to about 50 nucleotides complementary to the thioredoxin gene of the mammal under conditions such that the growth of the tumor is inhibited. The antisense oligonucleotide may be administered with a chemotherapeutic agent.
In another of its method aspects, this invention is directed to a method for inhibiting the growth of a mammalian tumor comprising, administering to a mammal suspected of having the tumor an effective amount of an antisense oligonucleotide comprising from about 3 nucleotides to about 50 nucleotides complementary to the thioredoxin reductase gene of the mammal under conditions such that the growth of the tumor is inhibited. The antisense oligonucleotide may be administered with a chemotherapeutic agent.
In another of its method aspects, this invention is directed to a method for inhibiting the metastasis of a mammalian tumor comprising, administering to a mammal suspected of having a metastatic tumor an effective amount of an antisense oligonucleotide comprising from about 3 nucleotides to about 50 nucleotides complementary to the thioredoxin gene of the mammal under conditions such that the metastasis of the tumor is inhibited. The antisense oligonucleotide may be administered with a chemotherapeutic agent.
In another of its method aspects, this invention is directed to a method for inhibiting the metastasis of a mammalian tumor comprising, administering to a mammal suspected of having the tumor an effective amount of an antisense oligonucleotide comprising from about 3 nucleotides to about 50 nucleotides complementary to the thioredoxin reductase gene of the mammal under conditions such that the metastasis of the tumor is inhibited. The antisense oligonucleotide may be administered with a chemotherapeutic agent.